Fermentative production of oxytetracycline by a new species of streptomyces



United States Patent 0 FERMENTATIVE PRODUCTION OF OXYTETRA- CYCLINE BY A NEW SPECIES OF STREPTO- MYCES Thomas H. Stoudt, Westfield, and Fred Tausig, Linden, N.J., assignors to Merck & Co'., Inc, Railway, .J., a corporation of New Jersey No Drawing. Filed Jan. 8, 1962, Ser. No. 164,984

4 Claims. (Cl. 19580) 2 the detailed description of this invention hereinafter provided.

In accordance with the present invention, it is now found that oxytetracycline is produced by growing a previously undescribed species of Streptomyces, Streptomyces sp. MA-590, in suitable nutrient mediums under aerobic conditions. This species was isolated from a sample of soil collected in Rahway, New Jersey. This new microorganism has been designated Streptomyces sp. MA-590 in the culture collection of the Merck Sharp & Dohme Research Laboratories at Rahway, New Jersey. A culture thereof has been deposited with the Fermentation Section of the Northern Utilization Research Branch, United States Department of Agriculture, at Peoria, Illinois, and added to its permanent culture collection as NRRL 2963.

The new microorganism is readily distinguished from the oxytetracyclineproducing culture S. Rimosus and S. platensis as will be readily apparent from the following table showing the morphological and cultural characteristics of the three species:

Streptomycee rimosue Description from Medium Bergeys Manual of Determinative Streptomyces sp. Mfr-590 Streptomyces platensz's NRRL-2364 Bacteriology, 7th Edition Gelatin Aerial mycelinm white. No soluble Aerialmycelium andsporesscantwhde Very slow liquefaction starting aft yellow pigment. No liquefaction. to light gray. No soluble pigment. about 12 days and still incomplete Liquefaction 50% in 14 days. after 30 days. Nutrient agar No aerial mycelium, poor growth. Heavy growth of aerial mycehum and No aerial mycelium and spores, substrat Faint yellowish pigment. spores, Mist color (Plates 53-113 Redpoor. Reverse Cream Yellow (R) t dish White M and P). Reverse Citrme Drab (R). Slight br w brown. soluble pigment. C apeks synthetic No aerial mycelinm, very limited Aerial mycelium and spores colorless, Mycehum Pale Smok Gray (R) d agar.

Asparagine agar Starch agar Glucose agar growth which is colorless and submerged. No soluble pigment.

Aerial mycelium white to pallid Quaker drab. Faint yellow soluble pigment.

Aerial mycelium limited; poor, thin growth. Colonies cinnamon-drab.

Aerial mycelium mouse-gray; growth fair growth. Very little submerged mycelium. N o soluble pigment.

Abundant aerial mycelium and spores, mouse-gray, good growth. Margin of colonies white. Reverse light gray. No soluble pigment.

Heavy aerial mycelium and spores; limited growth in substrate. Downy colonies light gray to dark gray. Reverse dark Brown. No soluble pig- Inent.

Aerial myceliurn and spores light gray to with dry, cracked surface. Yellowishdark gray. olomes with smooth brown pigment. powdery surface. Dark brown soluble pigment.

Milk Aerial mycelium grayislrwhite; thick Scant growth with no ring at surface. pellicle. Notpeptoruzed. No change Complete peptomzation in 14 days. in pH. N0 soluble pigment. pH alkaline.

Potato Aerial mycclium White to dark. Mod- Vegetative mycelium and spores light crate, wrinkled growth. Ycllowishgray; Good growth; surface wrinkled brown pigment. and downy. Reverse of single colonies very dark brown. Very dark brown soluble pigment.

Calcium malate agar (No record) No aerial mycelium or spores; light brown growth. Little growth in substrate in 14 days. Very small zone of calcium digestion, or none, in 14 days incubation. No soluble pigment.

Cellulose Not decomposed Slight growth. Cellulose not decomposed in 3 weeks.

Litmus milk (No record) Scant growth; small ring on surface.

Complete peptonization in 14 days. Reaction alkaline.

Nitrites Activity produced from nitrates 011 organic and synthetic media, nitrates not; reduced to nitrites after 2, 3, and 4 days of incubation.

ing to Pale Mouse Gray (R) with tufts of whlte growth rising above the background. Reverse Deep Olive Butt SR) becoming Dark Olive Bufi (R). o soluble pigment.

Moderate aerial myceliurn and spores. Mycehum white during early growth, later irregularly pigmented ranging from Light Grayish Olive (R) to Chaetura Black (R). Reverse Pale Ochraceous Buff (R) becoming Tawny (R). N 0 or very slight; brown soluble pigment.

Heavy aerial mycelium and spores. Mycelium. White becoming Pale Mouse Gray (R) with areas of black where growth heaviest. Reverse Cartridge Buff (R) to Cream Bufi (R). No soluble pigment.

Aerial mycclium and spores moderate to good. Myoelium White to Pale Mouse Gray (R) becoming almost white. Reverse Chamois (R) becoming Honey Yellow (R). Yellowishbrown soluble pigment.

(No record) Very good aerial mycelium and spores,

bite to Pale Mouse Gray (R); substrate spreading, wrinklcd. Brown soluble pigment.

Heavy growth of aerial rnycelium and spores. Mycelium Pale: Quaker Drab (R) with patches of closely related colors as well as areas of black and white. Black pigment in areas of best growth. Reverse Pale Oehraceous Salmon (R) becoming Cinnamon Buff (R) to Clay Color (R). Very slight greenlshyellow soluble pigment.

No aerial mycelium and spores. Substrata barely visible microscopically. No soluble pigment.

Scant growth forming partial ring at surface. No coagulation or hydrolysis in 14 days; slow coagulation and peptonization occur in 30 days. Slight alkaline reaction.

(No record).

Streptomyces rimosus Description from Medium Bergeys Manual of Determinative Streptomyces sp. MA590 Streptomyres plalensis NRRL-2864 Bacteriology, 7th Edition Casein Rapidly hydrolyzerL. Do.

Not hydrolyzed Hydrolyzed. Tyrosine agar No growth after days of 111cubat1on (No record). Hydrogen sulfide test. Good growth. Medium not d1sc0lored. Do.

Reverse dark brown. V. P.'test Negative Do. Emerson's agar Dark gray spores. Very dark brown Do.

soluble pigment. Bennett's agar- Heavy growth of aerial myceliurn and Do.

Morphology spores; little growth in substrate. Reverse black.

Conidia spherical about 0.8 microns in Conidia ovoid, measuring 0.7 to 0.9 by

by 0.8 to 1.4 microns, spirals numerous. Aerial mycelium limited; Ochre colored in center, colonial-buff atedge. Vegetative growth: Flat, smooth colonies with irregular edge. Yellow pigment.

diameter. no spirals or hooked ends found. Mycelium branched, light gray to dark gray. Colonies powdery.

0.8 to 1.2 microns in loose to tight spirals. Mycelium branching, about 0.7 to 0.8 microns in diameter. Colonies slightly raised, powdery; margin entire; peripheral surface area white to gray in center.

Spores in straight chains;

somewhat raised,

The new microorganism differs from S. rimosus in liquefying gelatin after a relatively short period of incubation and in producing heavy growth of aerial mycelium and spores on nutrient 'agar, Czapeks agar and Bennetts agar. Further, whereas S. rimosus reduces nitrate to nitrites, the new microorganism does not do so after 2, 3 and 4 days of incubation in both organic and synthetic nitrate media.

Streptomyces sp. MIA-590 differs from S. platcnsis in producing heavy growth of aerial mycelium and spores on nutrient agar. It produces colorless aerial mycelium and spores on Czapeks agar, whereas S. platensisproduces mouse gray aerial mycelium and spores on the same culture medium. On calcium malate agar the new microorganism grows very poorly and does not produce soluble pigments, whereas S. piatensz's grows heavily producing a greenish yellow soluble pigment on this medium. S. sp. MA-590 completely peptonizes milk in 14 days while S. platensis does so very weakly in days. It does not hydrolyze starch whereas .5. platcnsis does so.

Morphologically, the new microorganism diifers from both Streptomyces rimosus and Szreptomyces platensis in producing spherical spores on straight chains. S. rimosus produces cylindrical spores on numerous spirals, and S. platensis produces ovoid spores in loose to tightly closed spirals.

The above description of our new microorganism produring 'oxytetracycline is given as illustrative of suitable strains of Streptomyces sp. MA-590 which can be used in the production of oxytetracycline, but it is to be understood that 'the present invention is not to be limited to organisms answering this particular description. The present invention also contemplates the use of other species of Streptomyces sp. MA-590 which are mutants of the described organism such as those obtained by natural selection, or those produced by mutating agents for example, X-ray irradiation, ultraviolet irradiation, nitrogen mustards and the like.

In accordance with a further embodiment of the present invention, oxytetracycline is produced by growing Streptomyces sp. MA-590 or a mutant thereof in suitable nutrient mediums under aerobic conditions. In general, the nutrient mediums containing assimilable sources of carbon and nitrogen which are used for the production of oxytetracycline by the other oxytetracycline-producing species are also suitable for use with this new strain.

The examples which follow are illustrative of the methods by which Streptomyces sp. MA-590 can be used for the production and isolation of oxytetracycline.

EXAMPLE 1 Production of Oxytctmcycline by Fermentation A water spore suspension of an agar slant of Streptomyces sp. MA-590 was inoculated into a 250 ml. Erlenmeyer shake-flask containing 50 ml. of the following sterile medium:

After 72 hours incubation at 28 C. on a rotary shaker, the growth is inoculated into a 2-liter Erlenmeyer shake-fiask containing 750 ml.of the same sterile medium. This is incubated for 72 hours on a rotary shaker and used to inoculate 160 liters of the same sterile medium in the seed fermenter. The inoculated medium is incubated for 72 hours with agitation and aeration, and 40 liters of the resulting broth used to inoculate 51'0 liters of the same sterile broth in another fermenter. The resulting inoculated medium was incubated for 96 hours with agitation and aeration. The fermentation broth so produced assayed 100 ,ug/ml. of oxytetracycline.

EXAMPLE 2 Recovery of Oxytetracycline One hundred fifteen gallons of a fermentation broth produced by growing Streptomyces sp. MA59O as described in Example 1 and assaying 80 ig/ml. of oxytetracycline was adjusted to pH 1.9 with 50% sulfuric acid. Forty lbs. of diatomaceous earth was added and the broth was filtered to yield 112 gal. of filtrate containing 65 ig/ml. of activity. The filter cake was slurried in 55 gal. of water at 40 C., adjusted to pH 1.8, stirred for 30 minutes, and filtered. The two filtrates were combined. The 167 gal. thus obtained assayed 54 ,ug./ml., and contained a total of 34.1 g. of oxytetracycline activity. To this combined filtrate was added 275 lbs. of sodium chloride and the clear solution extracted twice in a two stage extractor with 70 gal. of normal butanol. The combined butanol extracts gal., containing 34.4 g. oxytetracycline activity) were concentrated in vacuo at a maximum temperature of 35 C., to a final volume of 17.5 1. which solution assayed 2 mg./ml. of oxytetracycline.

This butanol solution was extracted twice with 9 l. of 1 molar disodium phosphate and once with 8 l. of 20% sodium chloride. The pH was adjusted to 9.2 with 30% sodium hydroxide when necessary, and the aqueous phases were passed through 8 l. of fresh butanol. The aqueous phases removed a total of 2.5 g. of activity.

The butanol phases were extracted with 4 1., 8 1., 6 l., 6 l., and 4 1. portions of distilled water, the pH being adjusted to 2.2 with 6 N hydrochloric acid. The total yields and relative purities were 0.6 g. (0.4 ig/mg), 12 g. (230 ,ug./mg.), 6 g. (185 ,ug./mg.), 6 g. (260 ,ug./mg) and 2 l. ug/mg.) respectively.

The second, third and fourth extracts were combined and concentrated under reduced pressure to a final volume of 1005 ml.

1000 ml. of the above solution was subjected to a countercurrent distribution in ten 25 l. separatory funnels, using equal volumes of wet, acidified but-anol (pH 2.5) and dilute hydrochloric acid (pH 2.5) saturated with butanol. Each of the aqueous and but-anol phases were checked for potency, and aqueous phases four through eight were selected. These were combined and concentrated under vacuum to a final volume of 550 ml.

250 ml. of the above concentrate, pH 1.0, was adjusted to pH 5.0 by the slow addition of sodium hydroxide. After stirring for 20 minutes at room temperature the pH was readjusted to 7.0, and stirring continued for an additional hour. The amorphous precipitate was filtered, Washed with some water, and sucked dry. The yield of crude base was 10.08 g.

The dry cake was slurried in 60 ml. methanol, and 12 ml. of a saturated solution of calcium chloride in methanol was added slowly wit-h stirring. After 30 minutes 1 g. of activated charcoal was added and the mixture was filtered after an additional 20 minutes. The carbon cake was washed with 5 ml. of a saturated CaCl -methanol solution and 10 ml. of 1.5 normal methanolic hydrochloric acid. To the combined filtrate and washes was slowly added 7 ml. of concentrated hydrochloric acid. Slow stirring was continued for an hour and a half, after which the crystals were collected on a filter funnel, washed with 10 ml. of 1.5 N methanolic hydrochloric acid and 10 ml. of anhydrous methanol, and air-dried. The yield of oxytetracycline hydrochloride was 5.18 g. having a potency of 905 gJmg. (97% pure).

A sample of the hydrochloride so obtained was recrystallized once from anhydrous methanol. 95 mg. of the pure, shiny crystals of hydrochloride thus obtained was dissolved in 5 ml. distilled water and the pH adjusted to 6.5 with sodium hydroxide. The crystals of tree base, oxytetracycline, which formed were collected on a filter funnel, Washed with some distilled water, and dried in vacuo at 56 C. to yield the anhydrous free base. This material had a potency of 990 ag/mg. It melted at 183 185 C., with decomposition, undepressed by an authentic sample of anhydrous oxytetracycline. Its infrared and ultraviolet spectra were identical with those of oxytetracycline in every respect.

Various changes and modifications of the invention can be made and, to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope of the appended claims.

What is claimed is:

1. A process for producing oxytetracycline which comprises .growing an oxytetracycline producing strain of Streptomyces sp. MA-590 (NRRL 2963) under aerobic conditions in a nutrient medium.

2. A process tor producing oxyte-tracycline which comprises growing Streptomyces sp. MA-590 (NRRL 2963) under aerobic conditions in a nutrient medium.

3. A process for producing oxytetracycline which comprises growing Streptomyces tsp. MA-590 (N-RRL 2963) in a nutrient medium under aerobic conditions until substantial antibacterial activity is imparted to said medium.

4. A process for producing oxytetracycline which comprises growing Streptomyces sp. MA-590 (NRRL 2963) in a nutrient medium containing assimilable sources of carbon and nitrogen until substantial antibacterial activity is imparted to said medium, and recovering oxytetracycli-ne from the resulting fermentation broth.

References Cited in the file of this patent UNITED STATES PATENTS 2,516,080 Sobin et al. July 18, 1950 

1. A PROCESS FOR PRODUCING OXYTETRACYCLINE WHICH COMPRISES GROWING AN OXYTETRACYCLNE PRODUCING STRAIN OF STREPTOMYCES SP. MA-590(NRRL 2963) UNDER AEROBIC CONDITIONS IN A NUTRIENT MEDIUM. 